Adynamic response to cold pain reflects dysautonomia in type 1 diabetes and polyneuropathy

Cardiac autonomic neuropathy (CAN), widely assessed by heart rate variability (HRV), is a common complication of long-term diabetes. We hypothesized that HRV dynamics during tonic cold pain in individuals with type 1 diabetes mellitus (T1DM) could potentially demask CAN. Forty-eight individuals with long-term T1DM and distal symmetrical polyneuropathy and 21 healthy controls were included. HRV measures were retrieved from 24-h electrocardiograms. Moreover, ultra-short-term HRV recordings were used to assess the dynamic response to the immersion of the hand into 2 °C cold water for 120 s. Compared to healthy, the T1DM group had expectedly lower 24-h HRV measures for most components (p < 0.01), indicating dysautonomia. In the T1DM group, exposure to cold pain caused diminished sympathetic (p < 0.001) and adynamic parasympathetic (p < 0.01) HRV responses. Furthermore, compared to healthy, cold pain exposure caused lower parasympathetic (RMSSD: 4% vs. 20%; p = 0.002) and sympathetic responses (LF: 11% vs. 73%; p = 0.044) in the T1MD group. QRISK3-scores are negatively correlated with HRV measures in 24-h and ultra-short-term recordings. In T1DM, an attenuated sympathovagal response was shown as convincingly adynamic parasympathetic responses and diminished sympathetic adaptability, causing chronometric heart rhythm and rigid neurocardiac regulation threatening homeostasis. The findings associate with an increased risk of cardiovascular disease, emphasizing clinical relevance.

Cardiac vagal tone and cardiac sensitivity to the baroreflex. Additionally, validated measures of CVT and CSB were recorded using a designated electronic device (Neuroscope, Medifit Instruments, Enfield, Essex, UK), providing real-time measures of efferent and afferent central neurocardiac regulation, e.g., parasympathetic brainstem influence of the heart (Neuroscope, Medifit Instruments, Enfield, Essex, UK) 13,34,36 . Finally, blood pressure was continuously measured using a blood pressure monitor (Omron M4, Hoofddorp, Netherlands).
Tonic cold pain. The participants were situated in a 60-degree supine position and instructed to breathe spontaneously. The participants immersed their left open hand into circulating cooled water (2.0 °C ± 0.3 °C) and maintained it for 120 s or earlier if intolerable pain was reached. Following tonic cold pain exposure, participants were asked to rate the pain and unpleasantness intensity on a modified rating scale, organized as an 11-point Likert scale, ranging from 0 to 10 (0 = no pain, 10 = worst imaginable pain).
To derive the dynamic changes in the neurocardiac regulation assessed as a sympathetic and parasympathetic response, HRV was recorded continuously, starting 4 min before (pre-testing), during (initial response), and up to 16 min after (sustained and recovery responses). Subsequently, these recordings were divided into ultra-short HRV recordings of 2-min epochs 8,37 . To characterize the dynamic physiological pattern, HRV was derived from each of these epochs. To improve the robustness of our pre-testing baseline, we used the mean of the two epochs representing pre-tests (four to two and two to zero min before immersion).
Statistics. Data management was carried out using Epidata Software® (The Epidata Association, Odense, Denmark), and statistical analysis was performed using Stata® (StataCorp LLC v. 17.0, Texas, USA). Data were tested for normality using Shapiro-Wilk and by visual inspection of histograms and Q-Q plots. Baseline characteristics and HRV measures are presented as the median and interquartile range (IQR). Differences between T1DM and the control group were compared using unpaired t-tests. Calculations of standard error, confidence intervals, and p-values were calculated using bootstrapping with 1000 replications to accommodate for violations in normality and correlation structure. Categorical data are presented as numbers in each group and compared using Fisher's exact test. Spearman's rank correlation was used to test for associations between QRISK3 and HRV recordings, CVT and CSB. Group differences in HRV, in response to tonic cold pain, were calculated using repeated measures-ANOVA and subsequentially by linear comparison of the measures. Pre-test values used in tonic cold pain were calculated as the average between two consecutive 2-min pre-test measurements, recorded 4 and 2 min before exposure. The main outcome was analyzed using crude differences and subsequently adjusted for age and BMI (adjusted differences) using regression analysis with bootstrapping with 1000 replications and reported as mean with confidence interval. Relative changes reported as percentage in HRV measures in response to tonic cold pain was calculated as the percentual differences between the pre-test and the 2 to 4-min epoch recordings. A p-value of < 0.05 was considered significant.
Ethics approval and consent to participate. All participants gave written informed consent before enrolment, and ethical approval was granted by The Scientific Ethics Committee, The North Denmark Region (N-20130077). The study was performed in accordance with the Declaration of Helsinki and the International Council for Harmonization's guidelines for Good Clinical Practice.

Results
Data of all 69 participants were assessed. Two participants in the T1DM group were excluded due to noisy HRV data (less than 80% of the recording met the quality criteria), and one participant was excluded due to repeated episodes of bradycardia. As for the tonic cold pain response, seven participants in both the T1DM group and www.nature.com/scientificreports/ healthy controls group had missing data (they withdrew their hand before 120 s), or HRV data was too noisy and were therefore excluded from the analysis. For comparison, the two groups were matched on sex, age, weight, smoking status, and BMI, see Table 1. Participants with T1DM and polyneuropathy had significantly slower nerve conduction tests, higher heart rate (HR), and systolic blood pressure (SBP) in both rest and during a controlled breathing regime. 90% had orthostatic hypotension as an indicator of CAN. The presence of polyneuropathy did not influence the objective small fiber response to heat or subjective pain ratings to cold pain; however, the magnitude of unpleasantness was lower in the T1DM group. All participants in the T1DM group had diabetic retinopathy, and 31% had an elevated UACR ratio. eGFR and LDL cholesterol were lower in the T1DM group than in healthy controls.
HRV based on 24-h recordings. Based on the 24-h recordings, both time-and frequency domain measures were lower in the T1DM group than in the healthy controls, indicating diabetes-induced dysautonomia (see Table 2). In the T1DM group, significant negative correlations were found between QRISK3 score and www.nature.com/scientificreports/ Complementary neurocardiac measures. CSB was lower in the T1DM group than in healthy controls. A tendency towards decreased CVT in the T1DM group was found, but this did not meet significancy (p = 0.059).
Dynamic HRV responses in the healthy controls group. Compared to RR-intervals in the pre-testing period, the mean RR decreased for all time points following tonic cold pain (p < 0.047). In contrast, SDNN (p = 0.015) and RMSSD (p < 0.001) increased in the initial response to tonic cold pain exposure. Furthermore, LF (p = 0.031) and HF (p = 0.038) increased immediately after cold pain exposure compared to the pre-testing period. No significant changes were found for total power, VLF, and LF/HF. A notable late sympathetic shoot was found for LF/HF in the sustained response to tonic cold pain exposure.
Dynamic HRV responses in the T1DM group. When compared to RR-intervals in the pre-testing period, mean RR (p < 0.001) and RMSSD (p = 0.040) decreased as an initial response to tonic cold pain. Interestingly, this was accompanied by decreased LF (p = 0.004) and LF/HF ratio (p < 0.001). In the sustained response (6 to 8 min) (p = 0.015) and recovery phase (8 to 10 min) (p < 0.00), mean RR increased. Neurocardiac adaptability of the heart rate, reflected in the SDNN measure, was not evident until the recovery epochs at 10-12 min (p = 0.026) and 12-14 min (p = 0.005) after tonic cold pain. No significant changes were found for total power or HF. Differences in HRV responses to tonic cold pain between T1DM and healthy controls. Exposure to tonic cold pain revealed dynamic differences between healthy controls and individuals with T1DM and Table 2. Differences in HRV measures between T1DM and control group at rest (24-h), CVT, and CSB between T1DM and healthy controls, with and without adjustment for age and BMI. Healthy controls represent the standard, and negative values represent diminished activity in HRV measures in the T1DM group. Raw data values are reported as median (IQR). Values for crude and adjusted differences (*adjusted for age and BMI) are reported as mean (confidence interval) and performed using bootstrapping with 1000 replications. RMSSD root mean square of successive differences in NN intervals, SDNN standard deviation of NN intervals, SDNNI mean of the standard deviation of all NN intervals for each 5-min segment of a 24-h recording, SDANN standard deviation of all NN intervals for each 5-min segment of a 24-h recording, VLF very-low-frequency, LF low-frequency, HF high-frequency, LF/HF low-frequency/high-frequency ratio, CVT cardiac vagal tone, CSB cardiac sensitivity to the baroreflex. Significant p-values are reported in bold.

Discussion
In line with existing literature 29,30 , we found that HRV time-and frequency domain measures derived from 24-h ECG significantly discriminated our cohorts of individuals with T1DM and polyneuropathy and age-and sexmatched healthy controls, indicating diabetes-induced dysautonomia. We further investigated the neurocardiac adaptability by investigating the electrocardiographically dynamic responses representing HRV measures before, during, and after exposure to tonic cold pain. Compared to healthy, we found convincingly adynamic responses in the T1DM group, evident as a delayed increase in SDNN and decreased parasympathetic regulation, contributing to a more chronometric heart rhythm. The findings reveal impaired neurocardiac adaptability to rapidly adjust to potential threats (here mimicked with cold pain exposure), causing physical or psychological alterations, and consequently, maintained homeostasis is mistrusted.
Baseline characteristics. This study included 21 healthy and 48 individuals with T1DM and confirmed DSPN (decreased nerve conduction velocities and thermal pain tolerance), increased heart rate and systolic blood pressure, and 90% of had orthostatic hypotension, indicating both peripheral and severe autonomic nerve dysfunction. This was further supported by the mean clinical risk score QRISK3 of 20%, indicating a relatively high risk of cardiovascular events within the next 10 years. All participants in the T1DM group had diabetic retinopathy, and the number of participants with a UACR above > 30 mg/g (lower level of microalbuminuria) was larger in the T1DM group, reflecting the systemic presence of microvascular complications. Antihyperlipidemic and antihypertensive treatment is recommended in the diabetes treatment guidelines. This explains why  Table 3. SDNN Standard deviation of NN intervals, RMSSD root mean square of successive differences in NN intervals. www.nature.com/scientificreports/ www.nature.com/scientificreports/ a larger percentage of participants received these treatments in the T1DM group, reflected as lower levels of LDL than healthy.

Autonomic regulation reflected in HRV.
The HRV measures are an emergent property assessing the neurocardiac adaptability, reflecting the heart-brainstem interactions and regulated through the non-linear regulated autonomic nervous system 10 . HRV describes the fluctuations in the time intervals between consecutive heartbeats. Mean RR is simply the time of the inter-beat interval. SDNN is the standard deviation of the RR-intervals and is considered the most robust parameter to quantify the inter-beat variability and neurocardiac adaptability, and it reflects both sympathetic and parasympathetic modulation in response to physiological influences 38 . RMSSD and HF are strongly believed to represent central parasympathetic regulation 8 , in contrast to the LF component of HRV, which reflects both sympathetic and parasympathetic activity. However, the utility of the LF component of HRV is largely debated [39][40][41] . The LF/HF ratio is believed to reflect the sympathovagal balance or sympathetic modulations 8 . According to Shaffer et al., a low LF/HF reflects parasympathetic dominance, whereas a high LF/HF reflects sympathetic dominance 10 . Finally, the indices have proven valuable in assessing individual risk stratification for cardiovascular events and the degree of neurocardiac dysautonomia 6,42 . Our dynamic assessments provided the possibility to interpret the cold pain-induced comprehensive autonomic response, comparable to the assessment of orthostatic hemodynamics in response to, e.g., standing or tilting, representing initial, sustained, and recovery phases 43 . Compared to healthy, the diabetes group experienced less unpleasantness, but there was no difference in pain response to tonic cold pain or thermal heat stimulation. This rules out that peripheral neuropathies modulated the afferent upstream activation of the tonic pain experience, thereby biasing our HRV data. Consequently, as both groups were exposed to equipotent peripheral pain experiences, our findings of decreased parasympathetic response to tonic cold pain in the diabetic group are supported by impaired neurocardiac regulation, evident as a delayed increase in SDNN. Taken together, the adynamic HRV responses in T1DM to tonic pain indicate central dysautonomia, even when controlling for age and BMI.

Characteristics of HRV (ultra-short vs. long-term recordings).
We used ultra short-term HRV epochs (2 min) to assess the dynamic neurocardiac regulation. Such short epochs have less variability compared to 24-h recordings. Even though they are influenced by sinus arrhythmia, the baroreceptor reflex (negative feedback control of blood pressure), and rhythmic changes in vascular tone 8,37 , the 24-h recordings are further influenced by circadian rhythms, body core temperature, metabolism, sleep pattern, and the regulation of the renin-angiotensin system 8,37 , thereby providing the highest possible variability. The 24-h recordings are the gold standard for clinical HRV assessments because they are more accurate in predicting cardiovascular events than short recordings. Even though we use identical mathematical formulas to assess HRV from 24-h and shortterm recordings, they represent complementary physiological measures. Simplistically, RMSSD and HF have been suggested to quantify the portion of parasympathetic regulation and used to estimate the sympathovagal balance 8 . Such indices are often used to assess individual risk stratification for cardiovascular events and the degree of neurocardiac dysautonomia 42 .
An example is the LF/HF ratio, which may describe the sympathovagal balance under controlled conditions 44 . However, the measure should be interpreted cautiously in situations where the neurocardiac regulation is altered in response to external situations, such as the cold pressor test, primarily because the total power (sum of the energy in VLF, LF, and HF bands) is highly variable and changes in response to external stimuli that challenges homeostasis 10 . In that perspective, the short-term data reflect vulnerability to underreport differences. Nonetheless, the recordings were analyzed as a series of 2-min epochs and revealed significant differences providing robust and complementary information on the neurocardiac adaptability to cold pain exposure.

Healthy controls vs. T1DM in 24-h HRV recordings.
The 24-h recordings revealed impaired neurocardiac regulation, and the shown diminished RMSSD and HF support the diagnosis of cardiovascular autonomic neuropathy (CAN) characterized by severe sympathetic dominance and parasympathetic withdrawal. Even though HRV measures are known to be reduced in individuals with T1DM in comparison to healthy controls 9,28-31,45 , conflicting results exist, which may indicate co-activation or co-inhibition of the two tonically activated branches, allowing rapid physiological neurocardiac adaptation in response to the surroundings. Furthermore, attenuated HRV responses at rest, but not in response to cold pain, were shown in a cohort with 20 participants with type 2 diabetes compared to 10 healthy controls 33 . Consequently, LF/HF may not always index autonomic balance accurately 7,10,32,33 .
To our surprise, total power, VLF, LF, and HF components of the 24-h HRV measures were correlated to the mean QRISK3, supporting the relevance of assessing neurocardiac regulation with HRV measures. Interestingly, based on the ultra-short epochs, QRISK3 scores negatively correlated with SDNN, total power, VLF, LF, and HF between 2 and 6 min post-exposure, underlining that parasympathetic withdrawal and adynamic appearance of other HRV measures are associated with increased risk of having a cardiovascular event.
Complementary neurocardiac measures. The arterial baroreceptors buffer acute fluctuations in blood pressure during e.g., sympathetic dominance, deep breathing, or postural changes by transducing increased vascular distension into nervous electrical activity, which actuates parasympathetic activation and sympathetic inhibition 44 . Baroreceptor activity is activated through high-pressure (aortic arch) or both low-and high-pressure (carotid sinus) baroreceptors, triggering distinct pathways, the so-called cardio-vagal, the cardio-sympathetic or the vaso-sympathetic pathway 46 . We showed decreased CSB in T1DM, constituting the afferent branch of the cardiac-brainstem communication. Interestingly, we only found a tendency towards decreased CVT, con- www.nature.com/scientificreports/ stituting the efferent branch of the cardiac-brainstem communication, but previously it has been reported that CVT is reduced in T1DM 34 . Both findings are in accordance with the literature, plausibly explained by damage to central or peripheral (afferent and/or efferent) parts of the cardiac-brainstem circuit 47 .
Exposure to tonic cold pain. We found no group differences between healthy and T1DM in perceived pain, and hence, believe that the individuals were exposed to an equipotent painful stimulus, with a proportional autonomic response. Exposure to cold tonic pain is perceived with high inter-individual variability, which has classically been considered to activate the sympathetic response because it is supported by elevated adrenaline and noradrenaline levels, increased heart rate or blood pressure [13][14][15][17][18][19][20][21][22][23][24] . Nevertheless, novel data reveal that approximately 30% of healthy subjects respond to cold pain exposure with parasympathetic dominance 48 , suggesting a more complex response, where an individual physiological meaningful linear, and non-linear coactivation of the sympathetic and parasympathetic system is present.
HRV responses in healthy following to exposure to cold pain. We refined the existing methodologies and measured mean HRV responses in ultra-short epochs (2 min) before, during, and up to 14 min after cold tonic pain (2 min at 2 °C) to allow a more dynamic interpretation of the neurocardiac regulation in response to cold pain exposure. The experimental setting is comparable to, e.g., provocative tests such as treadmill tests, in which stressing the neurocardiac central regulatory system can reveal changes that otherwise may not be visible under resting conditions. In the healthy cohort, we show convincingly dynamic physiological adaptive capacity of the heart, evidenced by increased SDNN and RMSSD, supported by Sanchez-Gonzalez et al., who also found increased RMSSD in response to cold pain 49 16,17 . Furthermore, since especially parasympathetic modulation decreases with increasing age 50 , and as our cohort is approximately double the age as those in the Jarczewski et al. and MacArtney et al., the need for matched controls to reliably compare the HRV responses between our two groups is emphasized 16,17 .
The initial group level response to tonic cold pain showed an increase in total power and LF/HF ratio, indicating more LF (or less HF) power, plausibly revealing increased sympathetic response in response to cold pain exposure. The literature shows ambiguous results, some report increased LF 25,49 and HF power 25,49,51 within the first 2 min after hand immersion into ice water, whereas others describe decreased LF 17 and HF 52 , which may be explained by individual responses, different methodologies, different central brainstem regulations (co-inhibition or reciprocal activation of the two branches), sympathetic ceiling, withdrawal, or enhanced compensatory parasympathetic tone.
HRV responses in T1DM with polyneuropathy following exposure to cold pain. Throughout the entire recording in T1DM, SDNN did not differ from the pre-test epoch indicating chronometrical heart rhythm, regardless of tonic cold pain exposure. This is supported by decreased mean RR and RMSSD in the initial response to cold pain exposure, indicating rapid sympathetic dominance, which interestingly is characterized by decreased total power, LF and LF/HF ratio decreased, and unchanged HF. At first sight, one could speculate that it solely represents sympathetic dominance, but it could also indicate that parasympathetic tone is at a minimum, and that is why it cannot be modulated more-even by a provocative test. Taken together, it indicates altered central neurocardiac regulation and the presence of cardiovascular autonomic neuropathy 3,6 . As a late sustained response in the recovery phase, sympathetic withdrawal or relatively enhanced parasympathetic activity was shown by increased RR intervals, but most evident is the presence of adynamic responses in SDNN, RMSSD, and total power, indicating non-adaptability of the neurocardiac regulation. To our knowledge, no previous studies have investigated such dynamic HRV response to tonic cold pain exposure in T1DM, unmasking the complexity of the concomitant sympathetic and parasympathetic autonomic regulation.

Differences in HRV responses between groups.
In comparison to healthy, we found that time-domain (SDNN and RMSSD) and frequency-domain (total power, LF) responses to cold pain exposure were unphysiologically adynamic, leading to decreased cardiac adaptability, chronometrical HR, and ultimately diminished ability to counteract or adapt to threatening external factors. Interestingly, in response to tonic cold pain exposure, both T1DM and healthy controls experienced a rapid decrease in the LF/HF ratio; however, total power was only diminished in the T1DM group. As the parasympathetic response was unchanged, this finding indicates differences in sympathetic responses, which were less powerful and less adaptable in the T1DM group. This is further supported by a cross-sectional study conducted in a cohort of 52 participants with diabetes and 15 years of diabetes duration, where no alterations in HR and blood pressure were shown in response to cold pain exposure 7 . Taken together both branches show impaired responses. The parasympathetic response is adynamic and unadaptable, but the sympathetic response can still, but to a lesser degree than normal, be modulated.

Strengths and limitations.
This is the first time that the dynamic HRV response to tonic cold pain has been investigated in a cohort of individuals with T1DM and polyneuropathy and compared with an age-and sex-matched cohort of healthy controls. Unsurprisingly, we showed convincing differences in the HRV measures based on 24-h recordings, supporting dysautonomia in the T1DM group. Furthermore, we showed robust group differences in the capacity of adapting the neurocardiac regulation in responses to tonic cold pain exposure. However, the first obvious limitation is that the participants with T1DM have long-term diabetes and severe www.nature.com/scientificreports/ verified polyneuropathy and the majority have orthostatic hypotension, indicative of severe CAN. Thus, our findings support clinical findings, but unfortunately, cardiovascular autonomic reflex tests were not carried out to diagnose cardiovascular autonomic neuropathy, hampering the ability to stratify the cohort according to the CAN stage. Secondly, 2 min epochs are ultra-short, making them vulnerable to physiological noise such as sinus arrhythmia, respiration patterns, coughing, certain movements, or stress response caused by the cold pressor test. However, data was consistently calculated within meaningful predefined intervals, and HRV analyses were conducted by the same person by use of validated software, minimizing the inter-and intra-observer differences. Thirdly, including the Poincaré plot could have revealed non-linear dynamics of consecutive RR intervals and thus provided additional information on the heart rate variability changes, but this was not included in the study. Fourthly, it has been shown in healthy that the cold pressor test causes high inter-individual variability, reflected in primarily parasympathetic and sympathetic responses, and thus, group comparisons yield a source of error. Nevertheless, in comparison to healthy, we showed that the parasympathetic response in T1DM was convincingly adynamic, and the magnitude of the adaptability of the sympathetic response was decreased. Lastly, it could have strengthened the interpretation of the neurocardiac regulation if novel parameters of sympathetic and parasympathetic activation, such as periodic dynamic depolarization of the T-wave and deceleration capacity, were used, but these were not available in this study.

Conclusion
In T1DM, an attenuated sympathovagal response was shown as convincingly adynamic parasympathetic responses and diminished sympathetic adaptability, causing chronometric heart rhythm and rigid neurocardiac regulation threatening homeostasis. The findings associate with an increased risk of cardiovascular disease, emphasizing the clinical relevance. The method quantifies and illustrates the complex autonomic regulation where physiological meaningful linear and non-linear co-activation may unmask dysautonomia, ultimately in earlier and silent stages.

Data availability
The data used during the current study are not publicly available but are available from the corresponding author on reasonable request.